Making Cartilage from Umbilical Cord Stem Cells Without Growth Factors


Loïc Reppel and his colleagues at CNRS-Université de Lorraine in France have found that mesenchymal stem cells from human umbilical cord can not only be induced to make cartilage, but that these remarkable cells can make cartilage without the use of exogenous growth factors.

Mesenchymal stromal/stem cells from bone marrow (BM-MSC) have, for some time, been the “all stars” for cartilage regeneration. In fact, a very innovative clinic near Denver, CO has pioneered the use of BM-MSCs for patients with cartilage injuries. Chris Centeno, the mover and shaker, of this clinic has carefully documented the restoration of articular cartilage in many patients in peer-reviewed articles.

However, there is another “kid’ on the cartilage-regeneration block; mesenchymal stromal/stem cells from Wharton’s jelly (WJ-MSC). The advantages of these cells are their low immunogenicity and large cartilage-making potential. In this paper, which was published in Stem Cell Research and Therapy, Reppel and others evaluated the ability of WJ-MSCs to make cartilage in three-dimensional culture systems.

Reppell and his coworkers embedded WJ-MSCs isolated from the umbilical cords of new-born babies in alginate/hyaluronic acid hydrogel and grew them for over 28 days. These hydrogels were constructed by the spraying method. The hydrogel solution (for those who are interested, it was 1.5 % (m/v) alginate and hyaluronic acid (ratio 4:1) dissolved in 0.9 % NaCl) was sprayed an airbrush connected to a compressor. The solution was seeded with WJ-MSCs and then sprayed on a sterile glass plate. The hydrogel was made solid (gelation) in a CaCl 2 bath (102 mM for 10 minutes). Then small cylinders were cut (5 mm diameter and 2 mm thickness) with a biopsy punch. Then Reppel and others compared the chondrogenic differentiation of WJ-MSC in these three-dimensional scaffolds, without adding growth factors with BM-MSC.

Illustration of protocol steps used to perform scaffold construct and chondrogenic differentiation. After monolayer expansion, MSC were seeded at 3× 10 6 cells/mL of Alg/HA hydrogel. Hydrogel was sprayed, gelated, and cut into 5 mm diameter cylinders; scale bar = 5 mm. Scaffolds were cultivated in a 48-well plate in differentiation medium for 28 days. Alg/HA alginate/hyaluronic acid, MSC mesenchymal stromal/stem cells, P3 passage 3
Illustration of protocol steps used to perform scaffold construct and chondrogenic differentiation. After monolayer expansion, MSC were seeded at 3× 10 6 cells/mL of Alg/HA hydrogel. Hydrogel was sprayed, gelated, and cut into 5 mm diameter cylinders; scale bar = 5 mm. Scaffolds were cultivated in a 48-well plate in differentiation medium for 28 days. Alg/HA alginate/hyaluronic acid, MSC mesenchymal stromal/stem cells, P3 passage 3

After 3 days in culture, WJ-MSCs seemed nicely adapted to their new three-dimensional culture system without any detectable damage. From day 14 – 28, the proportion of WJ-MSC cells that expressed all kinds of cell surface proteins characteristic of MSCs (i.e., CD73, CD90, CD105, and CD166) decreased significantly. This suggests that these cells were differentiating into some other cell type.

After 28 days in this scaffold culture, both WJ-MSCs and BM-MSCs showed strong upregulation of cartilage-specific genes. However, WJ-MSCs exhibited greater type II collagen synthesis than BM-MSCs, and these differences were evident at the RNA and protein levels. Collagen II is a very important molecule when it comes to cartilage synthesis because chondrogenesis, otherwise known as cartilage production, occurs when MSCs differentiate into cartilage-making cells known as chondroblasts that begins secreting aggrecan and collagen type II that form the extracellular matrix that forms cartilage. Unfortunately, in order to complete the run to mature cartilage formation, the chrondrocytes must enlarge (hypertrophy), and express the transcription factor Runx2 and secrete collagen X. Unfortunately, WJ-MSCs expressed Runx2 and type X collagen at lower levels than BM-MSCs in this culture system.

Matrix synthesis detected after 28 days of chondrogenic induction. Proteoglycans and total collagen were stained by Alcian blue and Sirius red (a), respectively. To explore the synthesis of various collagens in depth, immunofluorescence (b) and immunohistochemistry staining (c) were performed and detected using fluorescence microscopy and light microscopy, respectively; scale bar = 100 μm. BM-MSC bone marrow-derived mesenchymal stromal/stem cells, WJ-MSC Wharton’s jelly-derived mesenchymal stromal/stem cells
Matrix synthesis detected after 28 days of chondrogenic induction. Proteoglycans and total collagen were stained by Alcian blue and Sirius red (a), respectively. To explore the synthesis of various collagens in depth, immunofluorescence (b) and immunohistochemistry staining (c) were performed and detected using fluorescence microscopy and light microscopy, respectively; scale bar = 100 μm. BM-MSC bone marrow-derived mesenchymal stromal/stem cells, WJ-MSC Wharton’s jelly-derived mesenchymal stromal/stem cells

These experiments only examined cells in culture, which is not the same as placing cells in a living animal, but it is a start. Thus, when they are seeded in the hydrogel scaffold, WJ-MSCs and BM-MSCs, after 4 weeks, were able to adapt to their environment and express specific cartilage-related genes and matrix proteins in the absence of growth factors. In order to properly make cartilage in clinical applications, WJ-MSCs must go the full way and express high levels of Runx2 and collagen X. However, these experiments show that WJ-MSCs, which in the past were medical waste, are a potential alternative source of stem cells for cartilage tissue engineering.

Reppel and his colleagues note in their paper that to improve cartilage production from WJ-MSCs, it might be important to mimic the physiological environment in which chondrocytes normally find themselves. For example, they could apply mechanical stress or even a low-oxygen culture system. Additionally, Reppel and others could apply stratified cartilage tissue engineering. Reppel thinks that they could adapt their spraying method to design new stratified engineered tissues by applying progressive cells and spraying hydrogel layers one at a time.

All in all, cartilage repair based with WJ-MSC embedded in Alginate/Hyaluronic Acid hydrogel will hopefully be tested in laboratory animals and then, perhaps, if all goes well, in clinical trials.

Scientists Grow New Diaphragm Tissue In Laboratory Animals


The diaphragm is a parachute-shaped muscle that separates the thoracic cavity from the abdominopelvic cavity and facilitates breathing. Contraction of the diaphragm increases the volume of the lungs, thus dropping the pressure in the lungs below the pressure of the surrounding air and causing air to rush into the lungs (inhalation). Relaxation of the diaphragm decreases the volume of the lungs and increases the pressure in the lungs so that it exceeds the pressure of the air, and air leaves the lungs (exhalation). The diaphragm is also important for swallowing. One in 2,500 babies are born with malformations or perforations in their diaphragms, and this condition is usually fatal.

The usual treatment for this condition involves the construction of an artificial patch that properly covers the lesion, but has no ability to grow with the infant and is not composed of contractile tissue. Therefore, it does not assist in contraction of the diaphragm to assist in breathing.

A new study might change the prospects for these newborn babies. Tissue engineering teams from laboratories in Sweden, Russia and the United States have successfully grown new diaphragm tissue in rats by applying a mixture of stem cells embedded in a 3D scaffold made from donated diaphragm tissue. Transplantation of this stem cell/diaphragm matrix concoction into rats allowed the animals to regrow new diaphragm tissue that possessed the same biological characteristics as diaphragm muscle.
The techniques designed by this study might provide the means for repairing defective diaphragms or even hearts.

Doris Taylor, who serves as the director of regenerative medicine research at the Texas Heart Institute and participated in this revolutionary study, said: “So far, attempts to grow and transplant such new tissues have been conducted in the relatively simple organs of the bladder, windpipe and esophagus. The diaphragm, with its need for constant muscle contraction and relaxation puts complex demands on any 3D scaffold. Until now, no one knew whether it would be possible to engineer.”

Paolo Macchiarini, the director of the Advanced Center for Regenerative Medicine and senior scientist at Karolinska Institutet, who also participated in this study, said: “This bioengineered muscle tissue is a truly exciting step in our journey towards regenerating whole and complex organs. You can see the muscle contracting and doing its job as well as any naturally grown tissue.”

To make their tissue engineered diaphragms, the team used diaphragm tissue that had been taken from donor rats. They stripped these diaphragms of all their cells, but maintained all the connective tissue. This removed anything in these diaphragms that might cause the immune systems of recipient animals to reject the implanted tissue, while at the same time keeping all the things that give the diaphragm its shape and form. In the laboratory, the decellularized diaphragms had lost all their elasticity. However, once these diaphragm matrices were seeded with bone marrow-derived stem cells and transplanted into recipient laboratory animals, the diaphragm scaffolds began to function as well as normal, undamaged diaphragms.

If this new technique can be successfully adapted to human patients, it could replace the damaged diaphragm tissue of the patient with tissue that would constantly contract and grow with the child. Additionally, the diaphragm could be repaired by utilizing a child’s own stem cells. As a bonus, this technique might also provide a new way to

Next, the team must test this technique on larger animals before it can be tested in a human clinical trial.

The study was published in the journal Biomaterials.

Porous Hydrogels Boost Stem Cell-Based Bone Production


Regenerative medicine relies upon the ability to isolate, manipulate, and exploit stem cells from our own bodies or from the bodies of stem cell donors. A present obstacle to present therapeutic strategies is the poor survival of implanted stem cells. There are also worries of about properly directing the differentiation of transplanted stem cells. After all, if implanted stem cells do not differentiate into the terminal cell types you want to be replaced, the use of such cells seems pointless.

To address this problem, David Mooney from the Wyss Institute and his colleagues have designed a three-dimensional system that might keep transplanted stem cells alive and happy, ready to heal.

Mooney’s group has adopted a strategy based on the concept of stem cell “niches.”. In our bodies, stem cells have particular places where they live. These stem cell-specific microenvironments provide unique support systems for stem cells and typically include extracellular matrix molecules to which stem cells attach.

Mooney and others have identified chemical and physical cues that act in concert to promote stem cell growth and survival. The chemical cues found in stem cell niches are relatively well-known but the physical and mechanical properties are less well understood at the present time.

Stem cells in places like bone, cartilage, or muscle, when cultured in the laboratory, display particular mechanical sensitivities and they must rest on a substrate with a defined elasticity and stiffness in order to proliferate and mature. As you might guess, reproducing the right physical properties in the laboratory is no mean feat. However, several laboratories have used hydrogels to generate the right combination of chemical and physical properties.

Mooney and his colleagues have made two hydrogels with very different properties. A stable, “bulk” gel is filled with small bubbles of a pore-making molecule called a “porogen,” which degrades quickly and leaves porous cavities in its wake. When the bulk hydrogel is combined with extracellular matrix molecules from stem cell niches and filled with tissue-specific stem cells, and the porogen, Mooney and his team can make an artificial bone-forming stem cell niche. The porous cavities in the hydrogel, in combination with the chemical signals, drive the stem cells to grow, and divide while expanding into the open spaces in the gel. Then the cell move from the hydrogel to form mineralized bone.

In small animal experiments, Mooney and his colleagues showed that a porous hydrogel with the correct chemical and elastic properties provides better bone regeneration than transplanting stem cells alone. The maturing stem cells deployed by the hydrogel also induce neighboring stem cell populations to contribute to the bone repair, which further amplifies their regenerative effects.

This study provides the first demonstration that adjusting the physical properties of a biomaterial can not only help deliver stem cells but also tune the behavior of those cells in a living organism. Even though Mooney has primarily focused on bone formation, he and his group believe that the hydrogel concept can be broadly applied to other regenerative process as well.

This work was published in Nature Materials 2015; DOI: 10.1038/nmat4407.

Biomaterial Sponge-Like Impant Traps Spreading Cancer Cells


Prof Lonnie Shea, from the Department of Biomedical Engineering at the University of Michigan and his team have designed a small sponge-like implant that has the ability to mop up cancer cells as they move through the body. This device has been tested in mice, but there is hope that the device could act as an early warning system in patients, alerting doctors to cancer spread. The sponge-like implant also seemed to stop rogue cancer cells from reaching other areas where they could establish the growth of new tumors. Shea and others published their findings in the journal Nature Communications.

According to Cancer Research UK, nine in 10 cancer deaths are caused by the disease-spreading to other areas of the body. Stopping the spread of cancer cells, or metastasis, is one of the ways to prevent cancers from becoming worse. Complicating this venture is the fact that cancer cells that circulate in the bloodstream are rare and difficult to detect.

Shea’s device is about 5mm or 0.2 inches in diameter and made of a “biomaterial” already approved for use in medical devices. So far, this implant has so far been tested in mice with breast cancer. Implantation experiments showed that it can be placed either in the abdominal fat or under the skin and that it tended to suck up cancer cells that had started to circulate in the body.

Cancer Cell Trapping Implants

The implant mimicked a process known as chemoattraction in which cells that have broken free from a tumor are attracted to other areas in the body by immune cells. Shea and others found that these immune cells are drawn to the implant where they “set up shop.” This is actually a natural reaction to any foreign body, and the presence of the immune cells also attracts the cancer cells to the implant.

Initially, Shea and others labeled cancer cells with fluorescent proteins that caused them to glow under certain lights, which allowed them to be easily spotted. However, they eventually went on to use a special imaging technique that can distinguish between cancerous and normal cells. They discovered that they could definitively detect cancer cells that had been caught within the implant.

Unexpectedly, when they measured cancer cells that had spread in mice with and without the implant, they showed that the implant not only captured circulating cancer cells, but it also reduced the numbers of cancer cells present at other sites in the body.

Shea, said that he and his team were planning the first clinical trials in humans fairly soon: “We need to see if metastatic cells will show up in the implant in humans like they did in the mice, and if it’s a safe procedure and that we can use the same imaging to detect cancer cells.”

Shea and his coworkers are continuing their work in animals to determine what the outcomes if the spread of the cancer spread was detected at a very early stage, which is something that is presently not yet fully understood.

Lucy Holmes, Cancer Research UK’s science information manager, said: “We urgently need new ways to stop cancer in its tracks. So far this implant approach has only been tested in mice, but it’s encouraging to see these results, which could one day play a role in stopping cancer spread in patients.”

Using Silk to Grow Salivary Glands in the Laboratory


Colloquially, we use the word “spit” to describe saliva, which is secreted by our salivary glands. Saliva is a complex combination of water, salts, proteins, small molecules, and other components that lubricate our throats and mouths to facilitate swallowing, coat our gums and teeth to maintain good gum health and keep tooth decay at bay, and keep our breath fresh. Insufficient salivation production increases tooth decay rates, causes chronic halitosis (bad breath), and can swallowing difficult. Additionally, there are no treatments for poorly-functioning salivary glands, and these glands have poor regenerative capabilities.

Patients who suffer from head/neck cancers and have been treated with radiation suffer from “xerostoma” or dry mouth. Certain medications can also cause dry mouth as can old age. 50% of older Americans suffer from xerostoma.

If that isn’t bad enough, salivary glands are notoriously hard to grow in the laboratory.  So they slow down when we grow old, do not regenerate and grow poorly, in at all, in the laboratory.  Is there any good news about salivary glands?

Make that a yes!  A research team from the University of Texas Health Science Center, led by Chih-Ko Yeh has discovered a process that may lead to the growth of salivary glands in cell culture.  Yeh and his team used purified silk fibers that had many of their contaminants removed to grow salivary stem cells from rat salivary glands.  These cells grew in the laboratory and after several weeks in culture, generated a three-dimensional matrix that covered the silk scaffolds and shared many characteristics with the salivary glands that grow in the mouth.

Yeh underscored the importance of this discovery: “Salivary gland stem cells are some of the most difficult cells to grow in culture and retain their function.”  This work in Yeh’s laboratory have is the first time that salivary gland stem cells have been grown in cell culture while retaining their salivary gland properties.

Yeh continued, “The unique culture system has great potential for future salivary gland research and for the development of new cell-based therapeutics.”

Silk, contrary to what you might think, is an excellent choice for stem cell scaffolding because it is natural, biodegradable, flexible, porous material that provides cells easy access to oxygen and nutrition.  Silk also does not cause inflammation, which is a problem with other types of stem cell scaffolds.

Since there are so few salivary gland stem cells in the human mouth, Yeh and his group plan to continue using the rat model to refine their techniques.  Eventually, Yeh and others would like to use stem cells derived from bone marrow or umbilical cord blood to regenerate salivary glands in human patients.

In fact, Yeh and his coworkers have pioneered protocols for harvesting large numbers of bone marrow stem cells from bone marrow and human umbilical cord blood and growing them in culture.  These stem cells are abundant and can be differentiated into different cell types by means of tissue engineering technologies.

Yeh hopes that by the next decade, human salivary stem cells or tissue engineered artificial salivary gland will be used to initiate salivary gland regeneration in human patients.

This research was published in Tissue Engineering part A 2015; 21(9-10).

3-D Printing to Make Replacement Body Parts


Advances in three-dimensional (3D) printing have produced a swell of interest in artificial organs that are designed to replace, or even enhance, human tissues.

3-D printed organs

At the Inside 3D Printing conference in New York on April 15–17, 2015, researchers from academia and industry are gathering to discuss the growing interest in using three-dimensional (3D) printing to make replacement body parts. Although surgeons are already using 3D-printed metal and plastic implants to replace bones, researchers are looking ahead to printing organs using cells as “ink.”  All the structures shown here were all 3D printed at Wake Forest Baptist Medical Center in Winston-Salem, North Carolina, and include a rudimentary proto-kidney (top left), complete with living cells.

Printed organs, such as a prototype outer ear that was developed by researchers at Princeton University in New Jersey and Johns Hopkins University in Baltimore, Maryland, will be featured at the conference.  This ear is printed from a range of materials: a hydrogel to form an ear-shaped scaffold, cells that will grow to form cartilage, and silver nanoparticles to form an antenna (see M.S. Mannoor et al. Nano Lett. 13, 2634−2639; 2013. This is just one example of the increasing versatility of 3D printing.

This New York meeting, which is being advertised as the largest event in the industry, will provide exposure for a whole world of devices and novelties. But it will also feature serious discussions on the emerging market for printed body parts.

The dream of bioprinting is to print organs that can be used for transplant. For example, at the Wake Forest Baptist Medical Center in Winston-Salem, North Carolina, researchers are developing a 3D-printed kidney. The project is in its early stages and the kidney is far from functional and some doubt that researchers will ever be able to print such a complex organ. Perhaps a more achievable near-term goal might be to print sheets of kidney tissue that could be grafted onto existing kidneys.

Printed replacement for skull

Printed structures made of hard metal or polymers are already on the market for people in need of an artificial hip, finger bone or facial reconstruction. This skull implant (grey) was made by Oxford Performance Materials of South Windsor, Connecticut, and was approved by US regulators in 2013. It is made of a polymer meant to encourage bone growth, to aid integration of the implant into the surrounding skeleton. The company also sells implants for facial reconstruction and for replacing small bones in the feet and hands.

3-D printed lung tree

One of the key advantages of using 3D printing for surgical implants is the opportunity to model the implant to fit the patient. This airway splint (shown on the right branch of the model trachea) was designed by researchers at the University of Michigan in Ann Arbor to fit an infant with a damaged airway. The splint was made out of a material that is gradually absorbed by the body as the airway heals. The research team benefited from the concentration of 3D-printing expertise that has built up in Michigan because of the US automobile industry, which uses the technology for printing prototypes and design samples.

The business of 3-D printing also includes titanium replacement hip joints, which can be tailored to fit individual people, and made-to-order polymer bones to reconstruct damaged skulls and fingers. Printed body parts brought in US $537 million last year, up about 30% on the previous year, says Terry Wohlers, president of Wohlers Associates, a business consultancy firm in Fort Collins, Colorado, that specializes in 3D printing.

3-D printed prosthetics

3D printing can also be used to generate cheap — and creative — prostheses.  A prosthetic hand can cost thousands of dollars, which is a burdensome expense when fitting it to a growing child.  Jon Schull founded a company called e-NABLE that provides free printed prosthetics to those in need, harnessing the efforts of hundreds of volunteers who own consumer-grade 3D printers. “When people get tired of printing Star Wars figurines, they give us a call,” he says.  The cost of materials for a printed prosthesis is about US $35.

3-D animal prosthesis

Also, 3-D printed prostheses are not just for humans.  For example, a duck named Buttercup was born with its left foot turned backwards.  The Feathered Angels Waterfowl Sanctuary in Arlington, Tennessee, arranged for the fowl to receive a new foot, complete with a bendable ankle.  Also in the an eagle, a box turtle and a handful of dogs also have been fitted with 3-D printed prostheses.

Scientists are looking ahead to radical emerging technologies that use live cells as ‘ink’, assembling them layer-by-layer into rudimentary tissues, says Jennifer Lewis, a bioengineer at Harvard University in Cambridge, Massachusetts. Bioprinting firm Organovo of San Diego, California, already sells such tissues to researchers aiming to test experimental drugs for toxicity to liver cells. The company’s next step will be to provide printed tissue patches to repair damaged livers in humans, says Organovo’s chief executive, Keith Murphy.

Lewis hesitates to say that 3D printing will ever yield whole organs to relieve the shortage of kidneys and livers available for transplant. “I would love for that to be true,” she says. “But these are highly complicated architectures.”

Bone Marrow Stem Cells and Tissue Engineering Give a Woman a New Smile


Massive injuries to the face can cause bone loss and “tooth avulsion.” Medically speaking, avulsion simply refers to the detachment of a body structure from its normal location by means of surgery or trauma. Dental implants and help with lost teeth, but if the facial bone has suffered so much loss that you cannot place implants in them, then you are out of luck. Dental prostheses can help, but these do not always fit very well.

Darnell Kaigler and his group at the University of Michigan Center for Oral Health wanted to help a 45-year-old woman who had lost seven teeth and a good portion of her upper jaw bone (maxilla) as a result of massive trauma to the face. This poor lady had some dentures that did not fit well and a mouth that did not work well.

Bone can be grown from stem cells, but getting those stem cells to survive and do what you want them to do is the challenge of regenerative medicine. Therefore, Dr. Kraigler and his group used a new technique to help this young lady, and their results are reported in the December 2014 issue of the journal Stem Cells Translational Medicine.

First, Kraigler and his co-workers extracted bone marrow stem cells from a bone marrow aspiration that was taken from the upper part of the hip bone (the posterior crest of the ilium for those who are interested).  They used a product called ixmyelocel-T from Aastrom Biosciences in Ann Arbor , MI. This product is a patient-specific, expanded multicellular therapy, cell-processing system that selectively expands mesenchymal cells, monocytes and alternatively activated macrophages, up to several hundred times more than the number found in the patient’s bone marrow, while retaining many of the hematopoietic cells collected from only a small sample (50ml) of the patient’s bone marrow. Thus, the healing cells from the bone marrow are grown and made healthy, after which the cells were bagged and frozen for later use.

ixmyelocel-T

Then the patient was readied for the procedure by having the gum tissue cut and lifted as a flap of tissue (under anesthesia, or course). Then four holes were drilled into the bone and setting screws were inserted. This is an important procedure, because implanted stem cells will not survive unless they have blood vessels that can bring them oxygen and nutrients. By drilling these holes, the tissue responds by making new blood vessels. To this exposed surface, the bone marrow-derived stem cells were applied with a tricalcium phosphate (TCP). TCP is a salt that will induce mesenchymal stem cells to form bone. Once the TCP + stem cell mixture was applied to the gum, a collagen membrane was placed over it, and the gum was then sewn shut with sutures.

Cell transplantation procedure. Front view (A) and top view (B) of the initial clinical presentation showing severe hard and soft tissue alveolar ridge defects of the upper jaw. Following elevation of a full-thickness gingival flap, the images show front view (C) and top view (D) of the severely deficient alveolar ridge, clinically measuring a width of only 2–4 mm. Front view (E) and top view (F) of the placement of “tenting” screws in preparation of the bony site to receive the graft. Placement of the β-tricalcium phosphate (seeded with the cells 30 minutes prior to placement at room temperature) into the defect (G), with additional application of the cell suspension following placement of the graft in the recipient site (H). Placement of a resorbable barrier membrane (I) to stabilize and contain the graft within the recipient site, and top view (J) of primary closure of the flap.
Cell transplantation procedure. Front view (A) and top view (B) of the initial clinical presentation showing severe hard and soft tissue alveolar ridge defects of the upper jaw. Following elevation of a full-thickness gingival flap, the images show front view (C) and top view (D) of the severely deficient alveolar ridge, clinically measuring a width of only 2–4 mm. Front view (E) and top view (F) of the placement of “tenting” screws in preparation of the bony site to receive the graft. Placement of the β-tricalcium phosphate (seeded with the cells 30 minutes prior to placement at room temperature) into the defect (G), with additional application of the cell suspension following placement of the graft in the recipient site (H). Placement of a resorbable barrier membrane (I) to stabilize and contain the graft within the recipient site, and top view (J) of primary closure of the flap.

Four months later, the patient underwent a cone-beam computed tomography (CBCT) scan. The bone regrowth can be seen in the figure below.

Cone-beam computed tomography (CBCT) scans. CBCT scans were used to render three-dimensional reconstructions of the anterior segment of the upper jaw and cross-sectional (top view) radiographic images to show volumetric changes of the upper jaw at three time points. (A, B): The initial clinical presentation shows 75% jawbone width deficiency. (C, D): Immediately following cell therapy grafting, there is full restoration of jawbone width. (E, F): Images show 25% resorption of graft at 4 months and overall net 80% regeneration of the original ridge-width deficiency.
Cone-beam computed tomography (CBCT) scans. CBCT scans were used to render three-dimensional reconstructions of the anterior segment of the upper jaw and cross-sectional (top view) radiographic images to show volumetric changes of the upper jaw at three time points. (A, B): The initial clinical presentation shows 75% jawbone width deficiency. (C, D): Immediately following cell therapy grafting, there is full restoration of jawbone width. (E, F): Images show 25% resorption of graft at 4 months and overall net 80% regeneration of the original ridge-width deficiency.

According to the paper, there was an “80% regeneration of the original jawbone.”

Into this newly regenerated bone, permanent dental implants were placed. The results are shown below.

Complete oral rehabilitation. Clinical presentation of the patient prior to initiation of treatment (A) and following completed oral reconstruction (B). (C): Periapical radiographs of oral implants showing osseointegration of implants and stable bone levels at the time of placement, 6 months following placement, and 6 months following functional restoration and biomechanical loading of implants with a dental prosthesis.
Complete oral rehabilitation. Clinical presentation of the patient prior to initiation of treatment (A) and following completed oral reconstruction (B). (C): Periapical radiographs of oral implants showing osseointegration of implants and stable bone levels at the time of placement, 6 months following placement, and 6 months following functional restoration and biomechanical loading of implants with a dental prosthesis.

Pardon me, but permit me an unprofessional moment when I say that this is really cool.  Of course, this patient will need to be observed over the next several years to determine the longevity of her bone regeneration, but the initial result is certainly something to be excited about.

Tricalcium phosphate or TCP has been used to induce the bone-making activities of mesenchymal stem cells.  It has also been used in several animal studies as a delivery vehicle for mesenchymal stem cells (for example, see Rai B, et al., Biomaterials 2010, 31:79607970; Krebsbach PH, et al., Transplantation 1997, 63:10591069; Zhou J, et al., Biomaterials 2010, 31:11711179).  TCP also seems to support stem cell proliferation, survival, and differentiation into bone.  Kresbach and others showed that TCP most consistently yielded bone formation when used as a delivery vehicle for mesenchymal stem cells compared to other biomaterials commonly used, such as gelatin sponges and demineralized bone matrix.  However, there are no studies that have ascertained how well stem cells attach to TCP, and this attachment is an important factor in determining how many stem cells reach the site of injury.  This study by Kaigler and his group (A. Rajan and others) showed that a 30-minute incubation of the cells with TCP gave sufficient attachment of the cells to the TCP for clinical use.  The efficiency of this incubation period was also not affected by the temperature.  

The other exciting features of this paper, is that most of the materials used in this study were commercially available.  The bone marrow stem cell isolation technique was pioneered by Dennis JE, and others in their 2007 article in the journal Stem Cells (25:25752582).  Effective commercialization of this technique has shown the efficacy of this procedure for clinical use.  This paper also shows the clinical feasibility of using TCP as a delivery vehicle for mesenchymal stem cell-based bone treatments.

In conclusion, I will quote the authors: “Cell survival and seeding efficiency in the context of tissue engineering and cell-therapy strategies are critical parameters for success that have not been rigorously examined in a clinical context. This study defined optimized conditions for these parameters using an autologous stem cell therapy to successfully treat a patient who had a debilitating craniofacial traumatic deficiency. To our knowledge, there have been no other clinical reports of cell therapy for the treatment of craniofacial trauma defects. This clinical report serves as solid foundation on which to develop more expanded studies using this approach for the treatment of larger numbers of patients with other debilitating conditions (e.g., congenital disorders) to further evaluate efficacy and feasibility.”

“In Body” Muscle Regeneration


Researchers at Wake Forest Baptist Medical Center’s Institute for Regenerative Medicine have hit upon a new strategy for tissue healing: mobilizing the body’s stem cells to the site of injury. Thus harnessing the body’s natural healing powers might make “in body” regeneration of muscle tissue is a possibility.

Sang Jin Lee, assistant professor of Medicine at Wake Forest, and his colleagues implanted small bits of biomaterial scaffolds into the legs of rats and mice. When they embedded these scaffolds with proteins that mobilize muscle stem cells (like insulin-like growth factor-1 or IGF-1), the stem cells migrated from the muscles to the bioscaffolds and formed muscle tissue.

“Working to leverage the body’s own regenerative properties, we designed a muscle-specific scaffolding system that can actively participate in functional tissue regeneration,” said Lee. “This is a proof-of-concept study that we hope can one day be applied to human patients.”

If patients have large sections of muscle removed because of infections, tumors or accidents, muscle grafts from other parts of the body are typically used to restore at least some of the missing muscle. Several laboratories are trying the grow muscle in the laboratory from muscle biopsies that can be then transplanted back into the patient. Growing muscle on scaffolds fashioned from biomaterials have also proven successful.

Lee’s technique overcomes some of the short-comings of these aforementioned procedures. As Lee put it, “Our aim was to bypass the challenges of both of these techniques and to demonstrate the mobilization of muscle cells to a target-specific site for muscle regeneration.”

Most tissues in our bodies contain a resident stem cell population that serves to regenerate the tissue as needed. Lee and his colleagues wanted to determine if these resident stem cells could be coaxed to move from the tissue or origin, muscle in this case, and embeds themselves in an implanted scaffold.

In their first experiments, Lee and his team implanted scaffolds into the leg muscles of rats. After retrieving them several weeks later, it was clear that the muscle stem cell population (muscle satellite cells) not only migrated into the scaffold, but other stem cell populations had also taken up residence in the scaffolds. These scaffolds were also contained an interspersed network of blood vessels only 4 weeks aster transplantation.

In their next experiments, Lee and others laced the scaffolds with different cocktails of proteins to boost the stem cell recruitment properties of the implanted scaffolds. The protein that showed the most robust stem cell recruitment ability was IGF-1. In fact, IGF-1-laced scaffolds had four times the number of cells as plain scaffolds and increased formation of muscle fibers.

“The protein [IGF-1] effectively promoted cell recruitment and accelerated muscle regeneration,” said Lee.

For their next project, Lee would like to test the ability of his scaffolds to promote muscle regeneration in larger laboratory animals.

Stem Cell Treatments to Repair Cartilage Defects in the Knee


Erosions of the cartilage that covers the surfaces at the ends of our leg bones has motivated several laboratories to undertake clinical studies to test new techniques to heal lost cartilage, particularly at the knee. Many of these techniques have their share of drawbacks and advantages, but the number of clinical trials to deal with cartilage lesions of the knee are increasing. Unfortunately, more work remains to be done, but much more is known about several of these techniques than before. This article will summarize many of these techniques.

Microfracture is a procedure in which several small holes are drilled into the end of the bone to enhance the migration of mesenchymal stem cells from the bone marrow to the site of the cartilage defect. These MSCs then differentiate into chondrocytes and make cartilage that fills the lesion with new cartilage. Unfortunately, the cartilage made in these cases is fibrocartilage and not hyaline cartilage. Fibrocartilage lacks the biomechanical strength and durability of hyaline cartilage and it typically deteriorates 18-24 months after surgery. When used to treat large lesions, 20-50% of all cases develop intralesional osteophytes and the sclerotic bone increases the failure rate of autologous chrondrocyte implantation 3-7X. Thus microfractionation is only performed under very specific conditions and only in young patients, since this technique does not work in older patients.

Microfracture

Autologous Chondrocyte Implantation or ACI uses a full-thickness punch biopsy from a low-weight-bearing region of the joint taken during an arthroscopic surgery. This biopsy contains chondrocytes that are grown in cell culture to a population of about 12-48 million chondrocytes, which are troweled into the lesion during a second arthroscopic surgery. Clinical trials have established that ACI is safe and effective for large knee lesions. Peterson and others and Minas and others have established that even after 10 years, patients who have been treated with ACI show good relief of pain and increased knee function.

In the Peterson study, questionnaires were sent to 341 patients. 224 of 341 patients replied to the questionnaires, and of these respondents, 74% of the patients reported their status as better or the same as the previous years 10-20 years after the procedure (mean, 12.8 years).  92% were satisfied and would have ACI again.  Knee function and pain levels were significantly better after the procedure than before.  From this study, Peterson and others concluded that ACI is an effective and durable solution for the treatment of large full-thickness cartilage and osteochondral lesions of the knee-joint, and that the clinical and functional outcomes remain high even 10 to 20 years after the implantation.

Minas and others analyzed data from 210 patients treated with ACI who were followed for more than 10 years. ACI provided durable outcomes with a survivorship of 71% at 10 years and improved function in 75% of patients with symptomatic cartilage defects of the knee at a minimum of 10 years after surgery. A history of prior marrow stimulation as well as the treatment of very large defects was associated with an increased risk of failure.
In comparison studies by Bentley and others, ACI produced superior results to mosaicplasty (osteochondral transplantation or cylinders of bone drilled form low-weight-bearing parts of the knee that are implanted in a mosaic fashion into the knee).  In the Bentley study, 10 of 58 ACI patients had failed grafts after 10 years, but 23 of 42 mosaicplasty patients had failed cartilage repair.  According to studies by Based and others, and Saris and others, ACI is also superior to microfractionation in the repair of large cartilage lesions (>3 cubic cm), but seems to provide the same outcomes as microfracture for smaller lesions, according to Knudsen and others.  There are drawbacks to ACI.  The tissue flap used to seal the cartilage implant sometimes becomes pathologically enlarged.  Other materials have been used to seal the patch, such as hyaluronic acid, or collagen types I and III, but the use of these materials increases the expense of the procedure and the likelihood that the immune system will response to the covering.  Also, ACI outcomes vary to such an extent that the procedure is simply too unstandardized at the present time to be used consistently in the clinic.

Autologous Cartilage Implantation

In an attempt to standardize ACI, several orthopedic surgeons have tried to add a supportive scaffold of some sort to the chondrocytes harvested from the patient’s body.  Several studies in tissue culture have shown that chondrocytes not only divide better, but also keep their identities as chondrocytes better in a three-dimensional matrix (see Grigolo et al, Biomaterials (2002) 23: 1187-1195 and Caron et al, Osteoarthritis Cartilage (2012) 20; 1170-1178).  Therefore, ACI has given way to MACI or Matrix-Induced Autologous Chondrocyte Implantation, which seeds the chondrocytes on an absorbable porcine-derived mixed collagen (type I and III) prior to implantation.  The implant is then secured into the debrided cartilage lesion by means of a fibrin cover.

Several case studies have shown that MACI has substantial promise, but individual case studies are the weakest evidence available.  To prove its superiority over ACI or microfracture surgery, MACI must be compared in controlled studies.  In the few studies that have been conducted, the superiority of MACI remains unproven to date.  Patients who received MACI or ACI showed similar clinical outcomes in two studies (Bartlett and others, Journal of Bone and Joint Surgery (2005) 87: 640-645; and Zeifang et al, American Journal of Sports Medicine (2010) 38: 924-933), although those who received MACI showed a significantly lower tendency for the graft to enlarge.  MACI is clearly superior to microfracture surgery (Basad, et al., Knee Surgery, Sports Traumatology and Arthroscopy (2010) 18: 519-527), but longer-term studies are needed to establish the superiority of MACI over other treatment options.

A slight variation of the MACI theme is to embed the chondrocytes in a gel-like material called hyaluronic acid (HA).  HA-embedded chondrocytes have been shown to promote the formation of hyaline cartilage in patients (Maracci et al., Clinical Orthopedics and Related Research (2005) 435: 96-105).  Even though the outcomes are superior for patients treated with HA-MACI, the recovery period is longer (Kon E, et al., American Journal of Sports Science (2011) 39: 2549-2567).  MACI is available in Europe but not the US to date.  FDA approval is supposedly pending.  Long-term follow-up studies are required to establish the efficacy of this procedure.

Future prospects for treating knee cartilage lesions include culturing collagen-seeded chondrocytes for a longer period of time than the three days normally used for MACI.  During these longer culture periods, the seeded chondrocytes mature, and make their own scaffolds, which ensure higher-quality cartilage and better chondrocyte engraftment (see Khan IM and others, European Cell Materials (2008) 16: 26-39).  Alternatively, joint cartilage responds to stress by undergoing cell proliferating and increasing in density.  This response is due to the production of growth factors such as Transforming Growth Factor-β1 and -β3 (TGF-β1 and TGF-β3).  This motivated some enterprising tissue engineers to use recombinant forms of these growth factors to grow cartilage in bioreactors under high-stress conditions.  Such a strategy has given rise to NeoCart, a tissue-engineered product that has gone through Phase I and II trials and has been shown in two-year follow-up studies to be safe and more effective than microfracture surgery (Crawford DC and others, Journal of Bone and Joint Surgery, American Volume. 2012 Jun 6;94(11):979-89 and Crawford DC, and others, Am J Sports Med. 2009 Jul;37(7):1334-43).

Mesenchymal stem cells (MSCs) from bone marrow and other sites have also been used to successfully treat cartilage lesions.  These types of treatments are less expensive than ACI and MACI, and do not require two surgeries as do ACI and MACI.  The studies that have been published using a patient’s own MSCs have been largely positive, although some pain associated with the site of the bone marrow aspiration is a minor side effect (see Centeno and others, Pain Physician (2008) 11:343-353; Emadedin, et al., Arch Iran Med (2012) 15: 422-428; Wong RL, et al., Arthroscopy (2013) 29: 2020-2028).  Fat-based MSCs have been tested as potential cartilage-healers in elderly patients (Koh YG, et al., Knee Surgery, Sports Traumatology, and Arthroscopy (Dec 2013, published on-line ahead of print date).  While these initial results look promising,, fat-based, MSCs have only just begun to be tested for their ability to regenerate cartilage.  Fat-based MSCs show different properties than their bone-marrow counterparts, and it is by no means guaranteed that fat-based MSCs can regenerate cartilage as well as MSCs from bone marrow.

Fresh cartilage grafts from donors (aka – cartilage allografts) use transplanted cartilage that has been freshly collected from a donor.  Fresh cartilage allografts have had positive benefits for young, active patients and the grafts have lasted 1-25 years (Gross AE, et al., Clinical Orthopedics and Related Research (2008) 466: 1863-1870).  Particulate cartilage allografts takes minced cartilage and lightly digests it with enzymes to liberate some of the cartilage-synthesizing chondrocytes, and then pats this material into the cartilage lesion, where it is secured with a fibrin glue plug.  The cartilage provides an excellent matrix for the synthesis of new cartilage, and the chondrocytes make new cartilage while seeded onto this cartilage scaffold.  Clinical experience with this technique includes a two-year follow-up in which MRI evidence showed good filling of the lesions (Bonner KF, Daner W, and Yao JQ, Journal of Knee Surgery 2010 23: 109-114 and Farr J, et al., Journal of Knee Surgery 2012 25: 23-29).  A variation on this technique uses a harvested hyaline cartilage plug that is glued into an absorbable scaffold before transplantation into the cartilage lesion.  This procedure had the same safety profile as microfracture surgery, but resulted in better clinical outcomes, high quality cartilage, and fewer adverse side effects (Cole JB et al., American Journal of Sports Medicine 2011 39: 1170-1179).  A clinical trial that tested this procedure remains uncompleted after the company suspended the trial because of conflicts with the FDA (Clinical Trial NCT00881023).

AMIC or Autologous Matrix-Induced Chondrogenesis is a cell-free treatment option in which the cartilage lesion is cleaned and filled subjected to microfracture, after which the lesion is filled with a mixed collagen matrix that is glued or stitched to the cartilage lesion.  The MSCs released by the microfracture procedure now move into a scaffold-laden cartilage lesion that induces the formation of hyaline cartilage.  This technique appears to aid the filling of full-thickness cartilage defects, and follow-up examinations have revealed that after 5 years, patients showed substantial improvements in knee function, pain relief and MRI analyses of knee cartilage showed high-quality cartilage in repaired lesion (Kusano T, et al., Knee Surgery, Sports Traumatology, and Arthroscopy 2012 20: 2109-2115; Gille J, et al., Archives of Orthopedic Trauma Surgery 2013 133: 87-93; Gille J, et al., Knee Surgery, Sports Traumatology, and Arthroscopy 2010 18: 1456-1464).

These are just a few of the new treatments of cartilage lesions of the knee and other joints.  As you can see, all of this will lead to greater repair of knee lesions and it is all being done without embryonic stem cells or destroying embryos.

Mesenchymal Stem Cells Repair Cartilage Defects in Cynomolgus Monkeys


Repairing cartilage defects in the knee represents one of the primary goals of orthopedic regenerative medicine. Cartilage that covers the joints, otherwise known as articular cartilage, has a limited capacity for repair, which leads to further degeneration of the cartilage when it is damaged if it remains untreated. A number of surgical options for treating cartilage defects include microfracture, osteochondral grafting, and cell-based techniques such as autologous chondrocyte implantation (ACI). Each of these procedures have been used in clinical settings. Unfortunately cartilage injuries treated with microfracturing deteriorate with time, since the cartilage made by microfracturing has a high proportion of softer. less durable fibrocartilage.  Also osteochondral grafting suffers from a lack of lateral integration between host and donor cartilage.

Alternatively, tissue engineering has shown some promise when it comes to the healing of cartilage defects.  Mesenchymal stem cells (MSCs) are multipotent progenitor cells that have the ability to differentiate into several different cell lineages including cartilage-making chondrocytes.  MSCs have theoretical advantages over implanted chondrocytes when it come to healing potential.  MSCs have the ability to proliferate without losing their ability to differentiate into mature chondrocytes and produce collagen II and aggrecan. In the short-term, bone marrow-derived MSCs combined with scaffolds have been successful in cartilage repair using animal models such as rabbits (Dashtdar H, et al., J Orthop Res 2011; 29: 1336-42) sheep (Zscharnack M, et al., Am J Sports Med 2010; 38: 185769) and horses (Wilke MM, et al.,l J Orthop Res 2007; 25: 9132).  

In a recent study, Kazumasa Ogasawara and Yoshitaka Matsusue and their colleagues from Shiga University of Medical Science in Shiga, Japan, tested the ability of expanded bone marrow-derived MSCs that had been placed in a collagen scaffold to improve healing of cartilage defects in cynomolgus macaques (type of monkey).  Before this study, there were no previous studies using MSCs from primates for cartilage repair.  The monkey MSCs were shown to properly differentiate into fat, bone, or cartilage in culture, and then were transplanted into the injured cartilage in the cynomolgus macaque.  The efficacy of these cells were ascertained at 6, 12, and 24 weeks after transplantation.

In culture, the cynomolgus MSCs were able to differentiate into fat, bone, and cartilage.

Characteristics of bone marrow-derived MSCs. Panel (a) demonstrates the colony-forming properties of MSCs isolated from bone marrow of cynomolgus macaques using the present protocol (arrows). Bar: 1 cm. Panel (b) shows the adipogenetic properties of MSC-derived cells from staining of lipid droplets with oil red O (arrowheads). Bar: 20 μm. Panel (c) confirms the osteoblastic properties of MSC-derived cells with alkaline phosphatase staining (arrowheads). Bar: 30 μm. Panel (d) confirms the chondrogenetic properties from immunostaining of type-II collagen. Type-II collagen-positive matrix is stained red. Bar: 0.5 mm. Read More: http://informahealthcare.com/doi/full/10.3109/17453674.2014.958807.
Characteristics of bone marrow-derived MSCs. Panel (a) demonstrates the colony-forming properties of MSCs isolated from bone marrow of cynomolgus macaques using the present protocol (arrows). Bar: 1 cm. Panel (b) shows the adipogenetic properties of MSC-derived cells from staining of lipid droplets with oil red O (arrowheads). Bar: 20 μm. Panel (c) confirms the osteoblastic properties of MSC-derived cells with alkaline phosphatase staining (arrowheads). Bar: 30 μm. Panel (d) confirms the chondrogenetic properties from immunostaining of type-II collagen. Type-II collagen-positive matrix is stained red. Bar: 0.5 mm.
Read More: http://informahealthcare.com/doi/full/10.3109/17453674.2014.958807.

Upon transplantation into cartilage defects in the knee cartilage of cynomolgus monkeys, MSCs were compared with collagen gel devoid of MSCs.  The knees that received the transplantations did not show any signs of irritation, bone spurs or infection.  All of the animals had so-called “full-thickness cartilage defects,” and those in the non-treated group showed cartilage defects that did not change all that much.  The cartilage defects of the gel group had sharp edges at 6 weeks that were thinly covered with reparative tissue by 12 weeks, and at 24 weeks, the defect was covered with thick tissue, but the central region of the defects often remained uncovered, with a hollow-like deformity.  In the cartilage defects of those animals treated with MSCs plus the collagen gel, the sharp edges of the defects were visible at 6 weeks after the operation, but at 12 weeks, the defects were evenly covered with yellowish reparative tissue.  At 24 weeks, the defects were covered with watery hyaline cartilage-like tissue that was very similar to the neighboring naïve cartilage.

Macroscopic observations of the repaired defects in the 3 groups at 6 weeks (a, d, g), 12 weeks (b, e, h), and 24 weeks (c, f, i) after implantation. Scale bar: 5 mm. Arrow in (d): the sharp edge of the defect is visible at 6 weeks in the gel group. Arrow in (f): a hollow-like deformity remains in the central region of the defect, despite thick coverage by the reparative tissue. Arrow in (g): the sharp edge of the defect is also visible in the MSC group at 6 weeks. Read More: http://informahealthcare.com/doi/full/10.3109/17453674.2014.958807.
Macroscopic observations of the repaired defects in the 3 groups at 6 weeks (a, d, g), 12 weeks (b, e, h), and 24 weeks (c, f, i) after implantation. Scale bar: 5 mm. Arrow in (d): the sharp edge of the defect is visible at 6 weeks in the gel group. Arrow in (f): a hollow-like deformity remains in the central region of the defect, despite thick coverage by the reparative tissue. Arrow in (g): the sharp edge of the defect is also visible in the MSC group at 6 weeks.
Read More: http://informahealthcare.com/doi/full/10.3109/17453674.2014.958807.

When evaluated at the tissue level, Ogasawara and Matsusue and others used a stain called toluidine blue to visualize the amount of cartilage made by each treatment.  As you can see in the picture below, the non-treated group didn’t do so well.  In the full-thickness defect the region below the cartilage was filled with amorphous stuff 6 weeks after the procedure, and at 12 weeks, amorphous stuff faintly stained with toluidine blue, which reflects the conversion of the amorphous stuff into bone.  At 24 weeks, bone tissue reappeared below the cartilage zone, even though the bone did not look all that normal (no trabecular structure but woven bone-like structure).

In the gel group, cartilage-like tissue is seen at 6 weeks, and at 12 weeks, the faintly stained layer covered the cartilage defect. At 24 weeks, the defect was covered with the cartilage-like stuff, even though the central region had only a little cartilage, as ascertained by toluidine blue staining.  The bone underneath the cartilage looked crummy and there was excessive growth of cartilage into the region underneath the cartilage layer.

In the MSC group, the bone underneath the cartilage healed normally, and at 12 weeks, the boundary between the articular cartilage and the bone layer beneath it had reappeared.  At 24 weeks, the thickness of the toluidine blue-stained cartilage layer was comparable to that of the neighboring naïve cartilage.

Even though the gel group showed most cartilage-rich tissue covering the defect, this was due to the formation of excessive cartilage extruding through the abnormal lower bone layer.  Despite the lower amount of new cartilage produced, the MSC group showed better-quality cartilage with a regular surface, seamless integration with neighboring naïve cartilage, and reconstruction of the bone underneath the cartilage layer.

Histological findings after toluidine blue staining in the 3 groups at 6 weeks (a, d, g), 12 weeks (b, e, h), and 24 weeks (c, f, i) after implantation. Scale bar: 2 mm. Dotted line in (a): amorphous reparative tissue filling the subchondral region. Arrowheads in (b): faint toluidine blue staining that reflects involvement of endochondral ossification. Arrowhead in (c): toluidine blue-negative reparative tissue covering the defect. Dotted line in (c): reconstructed subchondral bone consisting of woven bone-like structure. Arrowhead in (d): toluidine blue-positive cartilaginous tissue. Arrowhead in (e): thin faintly toluidine blue-positive layer covering the defect. Arrowhead in (f): the unstained central region of the cartilaginous layer covering the defect. Arrow in (f): excessive cartilage extruding through the deficient tidemark. Dotted line in (g): woven bone-like subchondral bone already re-appearing at 6 weeks. Arrowhead in (h): reconstructed tidemark distinctly discriminating the articular cartilage from the subchondral bone.
Histological findings after toluidine blue staining in the 3 groups at 6 weeks (a, d, g), 12 weeks (b, e, h), and 24 weeks (c, f, i) after implantation. Scale bar: 2 mm. Dotted line in (a): amorphous reparative tissue filling the subchondral region. Arrowheads in (b): faint toluidine blue staining that reflects involvement of endochondral ossification. Arrowhead in (c): toluidine blue-negative reparative tissue covering the defect. Dotted line in (c): reconstructed subchondral bone consisting of woven bone-like structure. Arrowhead in (d): toluidine blue-positive cartilaginous tissue. Arrowhead in (e): thin faintly toluidine blue-positive layer covering the defect. Arrowhead in (f): the unstained central region of the cartilaginous layer covering the defect. Arrow in (f): excessive cartilage extruding through the deficient tidemark. Dotted line in (g): woven bone-like subchondral bone already re-appearing at 6 weeks. Arrowhead in (h): reconstructed tidemark distinctly discriminating the articular cartilage from the subchondral bone.

This protocol has been nicely optimized by Ogasawara and Matsusue and their research team.  From these data, they conclude:  “Application in larger defects is certainly in line with future clinical use. If MSCs—under optimized conditions—turn out to be superior to chondrocyte implantation in experimental cartilage repair, the procedure should be introduced to clinical practice after well-controlled randomized clinical trials.”  Hopefully, clinical trials will commence before long.  This procedure uses a patient’s own MSCs, and if such a procedure could reduce or delay the number of knee replacements, then it would surely be a godsend to clinicians and patients alike.

Accelerating Bone Regeneration with Combination Gene Therapy and Novel Scaffolds


A truly remarkable paper in the journal Advanced Healthcare Materials by Fergal J. O’Brien and his co-workers from the Tissue Engineering Research Group at the Royal College of Surgeons in Dublin, Ireland has examined a unique way to greatly speed up bone regeneration.

Mesenchymal stem cells from bone marrow (other locations as well) can differentiate into bone-making cells (osteoblasts) that will make architecturally normal bone under particular conditions. The use of mesenchymal stem cells and a variety of manufactured biomaterial matrices and administered growth factors enhance bone formation by mesenchymal stem cells (M. Noelle Knight and Kurt D. Hankenson, Adv Wound Care 2013; 2(6): 306–316; also see Marx RE, Harrell DB. Int J Oral Maxillofac Implants 2014 29(2)e201-9; and Kaigler D, et al., Cell Transplant 2013;22(5):767-77).

Protein growth factors tend to have rather short half-lives when applied to growth scaffolds. A better way to apply growth factors is to use the genes for these growth factors and apply them to “gene activated scaffolds.” Gene-activated scaffolds consist of biomaterial scaffolds modified to act as depots for gene delivery while simultaneously offering structural support and a matrix for new tissue deposition. A gene-activated scaffold can therefore induce the body’s own cells to steadily produce specific proteins providing a much more efficient alternative.

In this paper by O’Brien and his groups, the genes for two growth factors, VEGF and BMP2, were applied to a gene-activated scaffold that consisted of collagen-nanohydroxyapatite. VEGF drives the formation of new blood vessels, and this fresh vascularization, coupled with increase bone deposition, which is induced by BMP2, accelerated bone repair.

Mind you, the assays in the paper were conducted in cell culture systems. However, O’Brien and his colleagues implanted these gene-activated scaffolds with their mesenchymal stem cells into rats that had large gaps in their skulls. In this animal model system for bone repair, stem cell-mediated bone production, in addition to increased blood vessel formation accelerated bone repair in these animals. Tissue examinations of the newly-formed bone showed that bone made from gene-activated scaffolds with mesenchymal stem cells embedded in them made thicker, more vascularized bone than the other types of strategies.

This is not a clinical trial, but this preclinical trial shows that vascularization and bone repair by host cells is enhanced by the use of nanohydroxyapatite vectors to deliver a combination of genes, thus markedly enhancing bone healing.

3D Printed Facial Implant Approved by the FDA


Three-dimensional printing uses modified ink-jet printers to spray cells and biomaterials into shapes that mimic human organs, tissues and structures. These three-dimensional printers have been used to make a variety of implantable structures.

Last year, Oxford Performance Materials announced that they had successfully created a 3D-printed implant that could replace 75 percent of a patient’s skull. This OsteoFab Patient Specific Cranial Device was made of PEKK (Polyetherketoneketone) biomedical polymer and was printed by using CAD files that had been developed to personally fit each patient’s specific dimensions. PEKK is an ultra high performance polymer used in biomedical implants and other highly demanding applications. The PEKK polymer has the advantage of being biomechanically similar to bone. The Osteofab skull implant was approved by the FDA in February of 2013.

Osteofab

The success of OsteoFab laid the groundwork for the recent FDA approval of Oxford’s OsteoFab Patient-Specific Facial Device, a customizable implant for facial reconstruction.

ORM 3D printed facial implant

Implants like this are known as “biocompatible implants,” which behave mechanically, in this case, like real bone.  The techniques developed by Oxford Performance Materials allow engineers to fabricate pieces that match an individual patient’s specific facial dimensions and structure in a manner that reduces the overall cost of the procedures required to surgically reconstruct a face after devastating injury. Due to these technical advances pioneered by Oxford Performance Materials, facial implants can be fabricated very quickly, which allows the plastic surgeons to get the patient into surgery sooner rather than later.

“With the clearance of our 3D printed facial device, we now have the ability to treat these extremely complex cases in a highly effective and economical way, printing patient-specific maxillofacial implants from individualized MRI or CT digital image files from the surgeon,” said Scott DeFelice, CEO of Oxford Performance Materials, in a statement. “This is a classic example of a paradigm shift in which technology advances to meet both the patient’s needs and the cost realities of the overall healthcare system.”

Oxford’s 3D-printed Osteofab cranial implants also have FDA approval and could potentially be combined with these facial implants into a single device for treating severe cases.  Although these facial implants have not yet been used in the United States, Oxford said the implants are now available to doctors and hospitals.

From artificial fingertips to airway splints that help babies breathe, 3D printing has provided the means to address complex surgical repairs.  The good news is that skull caps and facial bones are just the beginning of what 3D printing technologies can achieve.  We may soon see FDA approval for other bones, like knee caps, hips, and even small bones in the fingers and hands.

It’s all a part of a growing wave that could make 3D printers just as common as MRI machines in the tool kits used by physicians to repair and heal injured people.

Mesenchymal Stem Cells Make Tendons on Fabricated Collagen


Ozan Akkus and his colleagues from the Department of Mechanical and Aerospace Engineering at Case Western Reserve University in Cleveland, Ohio has succeeded in making fibers made completely from the protein collagen. Why is this a big deal? Because it is so bloody hard to do.

In a paper published the journal Advanced Functional Materials, Akkus and others describe the generation of their three-dimensional collagen threads. This is the first time anyone has described the formation of such threads made purely from collagen.

Collagen is a very widely distributed protein in our bodies. It is the major structural component of tendons, and most connective tissues, and as a whole, collagen composes approximately one-third of all the protein in our bodies. There are almost 30 different types of collagen; some collagens for stiff fibers and others form flat networks that act as cushions upon which cells and other tissues can sit.

Collagen biosynthesis is very complicated and occurs in several steps. First, the collagen genes are transcribed into messenger RNAs that are translated by ribosomes into collagen protein. However, collagen proteins are made in a longer, inactive form that must undergo several types of modifications before it is usable.

Collagen synthesis begins in a compartment of the cell known as the endoplasmic reticulum, which is a series of folded membranes associated with the nuclear membrane. Within the endoplasmic reticulum, the end piece of the collagen protein, known as the signal peptide, is removed by enzymes called signal peptidases that clip such caps off proteins. Now particular amino acids within the collagen protein chains are chemically modified. The significance of these modifications will become clear later, but two amino acids, lysine and proline, and -OH or hydroxyl groups added to them. This process is called “hydroxylation,” and vitamin C is an important co-factor for this reaction. Some of the hydroxylysine residues have sugars attached to them, and three collagen protein chains now self-associate to form a “triple ɣ helical structure.” This “procollagen” as it is called, is shipped to another compartment in the cell known as the Golgi apparatus. Within the Golgi apparatus, the procollagen it is prepared to be secreted to the cell exterior. Once secreted, collagen modification continues. Other proteins of the collagen protein chains called “registration peptides” are clipped off by procollagen peptidase to form “tropocollagen.” Multiple tropocollagen molecules are then lashed together by means of the enzyme lysyl oxidase, which links hydroxylysine and lysine residues together in order to form the collagen fibrils. Multiple collagen fibrils form a proper collagen fiber. Variations on a theme are also available, since collagen can also, alternatively, attached to cell membranes by means of several types of proteins, including fibronectin and integrin.

collagen1

Now, if the cells has to go through all that just to make a collagen fiber, how tough do you think it is to make collagen fiber in a culture dish? Answer – way hard. In order to make collagen threads, Akkus and his team had to use a novel method for mature collagen production, and then they compacted the collagen molecules by means of the mobility of these molecules in an electrical field. This “electrophoretic compaction” method also served to properly align the collagen molecules until they formed proper collagen threads. Biomechanical analyses of these fabricated collagen threads showed that they had the mechanical properties of a genuine tendon. Akkus’ group when one step further and showed that a device they designed with movable electrodes could fabricate continuous collagen threads (). Thus, Akkus and his crew showed that they could make as many collagen threads as they needed and that these threads worked like tendons (see here for video). Are these guys good or what?

A. Schematic of basic electro-chemical cell layout for collagen alignment; B. Polarized image confirming the alignment of ELAC; C. Human mesenchymal stem cells on ELAC threads at day 1 and day 14. Cell form a confluent layer on day 14. Scale bar: 0.5 mm.
A. Schematic of basic electro-chemical cell layout for collagen alignment; B. Polarized image confirming the alignment of ELAC; C. Human mesenchymal stem cells on ELAC threads at day 1 and day 14. Cell form a confluent layer on day 14. Scale bar: 0.5 mm.

Nest, Akkus and his gang seeded collagen threads with mesenchymal stem cells (MSCs) from bone marrow. Remarkably, these collagen thread-grown mesenchymal stem cells differentiated into tenocytes, which are the cells that made tendons. Normally, MSCs do not readily form tenocytes in the laboratory, and they do not easily make tendons. However, in this case, the MSCs not only differentiated into tenocytes and made tenocyte-specific proteins and genes, but they do so without the addition of exogenous growth factors; the collagen threads were all the cells needed.

The seeded MSCs made Collagen I, which is the most abundant collagen of the human body, and is present in scar tissue, tendons, skin, artery walls, corneas, the endomysium surrounding muscle fibers, fibrocartilage, and the organic part of bones and teeth. Other tendon-specific proteins that were made included tenomodulin, and COMP (Cartilage oligomeric matrix protein). Furthermore, the electrically-aligned collagen does a better job of inducing the tenocyte fate in MSCs than collagen that is randomly oriented.

These remarkable and fascinating results demonstrate scaffolds made of densely compacted collagen threads stimulates tendon formation by Mesenchymal stem cells. Thus electrically aligned collagen as a very promising candidate for functional repair of injured tendons and ligaments. Now it is time to show that this can work in a living creature. Let the preclinical trials commence!!

Orthopedic Regeneration With a Combination of Stem Cells, Gene Therapy, and Tissue Engineering


A Duke University research team has combined synthetic scaffolding materials with gene delivery techniques to generate replacement cartilage precisely where it’s needed in the body.

The ingenious strategy utilized by this research project circumvents the need for large quantities of growth factors, which are expensive and difficult to apply after implantation. The Duke team led by Farshid Guilak, director of orthopedic research at Duke University Medical Center, used gene therapy to make stem cells that synthesize their own growth factors.

In brief, Guilak and his collaborators used genetically engineered viruses to transfer genes to stem cells embedded in a synthetic matrix. Upon infection, the stem cells grew and differentiated as needed, but the scaffolding provided the necessary structural cues for the stem cells to move to the proper configuration and form cartilage with the proper shape and biomechanical properties.

Guilak has devoted several years to developing biodegradable synthetic scaffolds that mimic the mechanical properties of cartilage. After testing many different scaffolds, he settled on a 3D woven poly(ε-caprolactone) scaffold, which is completely biodegradable and provides an excellent structural matrix for the synthesis of cartilage.  However, an additional challenge for engineering good cartilage is to coax stem cells embed themselves in the scaffold while differentiating into cartilage-making cells, known as chondrocytes, after the scaffold has been implanted into a living organism.

One widely used strategy is to treat the stem cells with growth factors to induce chrondrocyte formation and cartilage production. Such cartilage can be implanted after it has been grown in the laboratory. However, this approach has some inherent limitations.

Guilak explained that “a major limitation in engineering tissue replacements has been the difficulty in delivering growth factors to the stem cells once they are implanted in the body.” Guilak continued: “There’s a limited amount of growth factor that you can put into the scaffolding, and once it’s released, it’s all gone. We need a method for long-term delivery of growth factors, and that’s where the gene therapy comes in.”

To tackle this perennial problem, Guilak tapped a talented colleague of his, Charles Gersbach, an assistant professor of biomedical engineering, who happens to also be a gene therapy expert.

Gersbach looked at the tissue engineering problem in an entirely new way and suggested that if the mountain will not come to Mohammed (that is to say if the growth factors cannot be given to stem cells after implantation), then Mohammed should grow his own mountain (the stem cells should be genetically engineered to make their own growth factors). Unfortunately, the conventional gene therapy methods are too complex to be commercially feasible. Typically, stem cells are collected, infected with genetically modified viruses that introduces new genes into them, grown to large numbers, and applied to synthetic cartilage scaffolds and implanted into the patient. Sounds like a headache? That’s because it is.

Fortunately, Gersbach had a slick gene therapy trick up his lab coat sleeve: “There are a few challenges with that process, one of them being that there are way too many extra steps,” said Gersbach. “So we turned to a technique I had previously developed that affixes the viruses that deliver the new genes onto a material’s surface.”

A microscopic view using electron microscopy of human stem cells and viral gene carriers adhering to the fibers of a polymer scaffold.  Photo source:  http://www.pratt.duke.edu/news/gene-therapy-might-grow-replacement-tissue-inside-body.
A microscopic view using electron microscopy of human stem cells and viral gene carriers adhering to the fibers of a polymer scaffold. Photo source: http://www.pratt.duke.edu/news/gene-therapy-might-grow-replacement-tissue-inside-body.

This new study combines Gersbach’s gene therapy technique—dubbed biomaterial-mediated gene delivery—to induce those human mesenchymal stem cells embedded in Guilak’s synthetic cartilage scaffolding to produce growth factor proteins (in particular a molecule called transforming growth factor β3  or TGF-β3). Based on the results of their experiments, the technique works and that the resulting synthetic, composite cartilage-like material is at least as good biochemically and biomechanically as if the growth factors were introduced in the laboratory.

“We want the new cartilage to form in and around the synthetic scaffold at a rate that can match or exceed the scaffold’s degradation,” said Jonathan Brunger, a graduate student who has spent time in both Guilak’s and Gersbach’s laboratories developing and testing the new technique. “So while the stem cells are making new tissue (in the body), the scaffold can withstand the load of the joint. In the ideal case, one would eventually end up with a viable cartilage tissue substitute replacing the synthetic material.”

This particular study examines cartilage regeneration, but Guilak and Gersbach hope that their technique could be applied to the regeneration of many different kinds of tissues, especially orthopaedic tissues such as tendons, ligaments and bones. Also, because the platform comes ready to use with any stem cell, it presents an important step toward commercialization.

“One of the advantages of our method is getting rid of the growth factor delivery, which is expensive and unstable, and replacing it with scaffolding functionalized with the viral gene carrier,” said Gersbach. “The virus-laden scaffolding could be mass-produced and just sitting in a clinic ready to go. We hope this gets us one step closer to a translatable product.”

Citation: “Scaffold-mediated lentiviral transduction for functional tissue engineering of cartilage.” Brunger, J.M., Huynh, N.P.T., Guenther, C.M., Perez-Pinera, P., Moutos, F.T., Sanchez-Adams, J., Gersbach C.A., and Guilak F. PNAS Plus, 2014. DOI: 10.1073/pnas.1321744111/-/DCSupplemental

Nanometer Scaffolds Regulate Neural Stem Cells


In the laboratory, stem cells can grow in liquid culture quite well in many cases, but this type of culture system, though convenient and rather inexpensive, does not recapitulate the milieu in which stem cells normally grow inside our bodies. Inside our bodies, stem cells stick to all kinds of surfaces and interact with and move over a host of complex molecules. Many of the molecules that stem cells contact have profound influences over their behaviors. Therefore, reconstituting or approximating these environments in the laboratory is important even though it is very difficult.

Fortunately nanotechnology is providing ways to build surfaces that approximate the kinds of surfaces stem cells encounter in our bodies. While this field is still in its infancy, stem cell-based nanotechnology may provide strategies to synthesize biologically relevant surfaces for stem cell growth, differentiation, and culture.

One recent contribution to this approach comes from Jihui Zhou and his team from the Fifth Hospital Affiliated to Qiqihar Medical University. Zhou and his co-workers prepared randomly oriented collagen nanofiber scaffolds by spinning them with an electronic device. Collagen is a long, fibrous protein that is found in tendons, ligaments, skin, basement membranes (the substratum upon which sheets of cells sit), bones, and is also abundant in cornea, blood vessels, cartilage, intervertebral disc, muscles, and the digestive tract. Collagen is extremely abundant in the human body; some 30% of all the proteins in our bodies are collagen. It is the main component in connective tissues.

There are many different types of collagen. Some types of collagen form fibers, while others for sheets. There are twenty-eight different types of collagen. Mutations in the genes that encode collagens cause several well-known genetic diseases. For example, mutations in collagen I cause osteogenesis imperfecta, the disease made famous by the Bruce Willis/Samuel T. Jackson movie, “Unbreakable.” Mutations in Collagen IV cause Alport syndrome, and mutations in either collagen III or V cause Ehlers-Danlos Syndrome.

Wen cells make fibrous collagen, they weave three collagen polypeptides together to form a triple helix protein that is also heavily crosslinked. This gives collagen its tremendous tensile strength.

Collagen fibers
Collagen fibers

In this experiment, electronic spinning technology made the collagen fibers and these fibers had a high swelling ratio when placed in water, high pore size, and very good mechanical properties.

Zhou grew neural stem cells from spinal cord on these nanofiber scaffolds and the proliferation of the neural stem cells was enhanced as was cell survival. Those genes that increase cell proliferation (cyclin D1 and cyclin-dependent kinase 2) were increased, as was those genes that prevent cells from dying (Bcl-2). Likewise, the expression of genes that cause cells to die (caspase-3 and Bax) decreased.

Thus novel nanofiber scaffolds could promote the proliferation of spinal cord-derived neural stem cells and inhibit programmed cell death without inducing differentiation of the stem cells. These scaffolds do this by inducing the expression of proliferation- and survival-promoting genes.

Artificial Bones From Umbilical Cord Stem Cells


I am back from vacation. We visited some colleges in Indiana for my daughter who will be a senior this year. She really liked Taylor University and Anderson University. We’ll see if the tuition exchange works out.

Now to blogging.

Scientists from Granada, Spain have patented a hew biomaterial that consists of activated carbon cloth that just happens to be able to support the growth of cells that have the ability to regenerate bone. These results came from experiments that were conducted outside any living animals, but they hope to confirm these results in a living animal in the near future.

This new biomaterial facilitates the growth of bone-making cells derived from umbilical cord stem cells. This activated carbon cloth acts as a scaffold for cells that differentiate into “osteoblasts,” which are bone-building cells. This activated carbon cloth gives the osteoblasts a proper surface upon which to promote the growth of new bone.

Bone loss as a result of cancer, trauma, or degenerative bone diseases requires replacement bone to heal to damaged bone. Making new bone in the laboratory that can be transplanted is an optimal strategy for treating these patients.

Even though this laboratory-made bone was not used in living laboratory animals to date, the laboratory results look quite impressive. In the future, such techniques could help manufacture medicines or other sources of material to repair bone or lost cartilage. Once such artificial bone has been made in the laboratory, the Spanish team hopes to transplant it into rats or rabbits to determine if it can regenerate bone in such creatures.

Presently, no materials exist to replace lost bone. The method used to make bone by the research team from Granada uses a three-dimensional support that facilitates the production of those cell types that regenerate bone without the need for additional growth factors.

The growth of these umbilical cord stem cells on activated carbon cloth produced a product that could produce organic bone, but also mineralize the organic bone matrix. This patent could have numerous clinical applications in regenerative medicine and the Granada group hopes to obtain funding to continue this work and achieve their ultimate objective: to regenerate bones by implanting biomaterial in patients with bone diseases.

Improving Cartilage Production By Stem Cells


To repair cartilage, surgeons typically take a piece of cartilage from another part of the injured joint and patch the damaged area, this procedure depends on damaging otherwise healthy cartilage. Also, such autotransplantation procedures are little protection against age-dependent cartilage degeneration.

There must be a better way. Bioengineers want to discover more innovative ways to grow cartilage from patient’s own stem cells. A new study from the University of Pennsylvania might make such a wish come true.

This research, comes from the laboratories of Associate professors Jason Burdick and Robert Mauck.

“The broad picture is trying to develop new therapies to replace cartilage tissue, starting with focal defects – things like sports injuries – and then hopefully moving toward surface replacement for cartilage degradation that comes with aging. Here, we’re trying to figure the right environment for adult stem cells to produce the best cartilage,” said Burdick.

Why use stem cells to make cartilage? Mauck explained, “As we age, the health and vitality of cartilage cells declines so the efficacy of any repair with adult chondrocytes is actually quite low. Stem cells, which retain this vital capacity, are therefore ideal.”

Burdick and his colleagues have long studied mesenchymal stem cells (MSCs), a type of adult stem cell found in bone marrow and many other tissues as well that can differentiate into bone, cartilage and fat. Burdick’s laboratory has been investigating the microenvironmental signals that direct MSCs to differentiate into chondrocytes (cartilage-making cells).

chondrocytes
chondrocytes

A recent paper from Burdick’s group investigated the right conditions for inducing fat cell or bone cell differentiation of MSCs while encapsulated in hydrogels, which are polymer networks that simulate some of the environmental conditions as which stem cells naturally grow (see Guvendiren M, Burdick JA. Curr Opin Biotechnol. 2013 Mar 29. pii: S0958-1669(13)00066-9. doi: 10.1016/j.copbio.2013.03.009). The first step in growing new cartilage is initiating cartilage production or chondrogenesis. To do this, you must convince the MSCs to differentiate into chondrocytes, the cells that make cartilage. Chondrocytes secrete the spongy matrix of collagen and acidic sugars that cushion joints. One challenge in promoting MSC differentiation into chondrocytes is that chondrocyte density in adult tissue is rather low. However, cartilage production requires that the chondrocytes be in rather close proximity.

Burdick explained: “In typical hydrogels used in cartilage tissue engineering, we’re spacing cells apart so they’re losing that initial signal and interaction. That’s when we started thinking about cadherins, which are molecules that these cells used to interact with each other, particularly at the point they first become chondrocytes.”

Desmosomes can be visualized as rivets through the plasma membrane of adjacent cells. Intermediate filaments composed of keratin or desmin are attached to membrane-associated attachment proteins that form a dense plaque on the cytoplasmic face of the membrane. Cadherin molecules form the actual anchor by attaching to the cytoplasmic plaque, extending through the membrane and binding strongly to cadherins coming through the membrane of the adjacent cell.
Desmosomes can be visualized as rivets through the plasma membrane of adjacent cells. Intermediate filaments composed of keratin or desmin are attached to membrane-associated attachment proteins that form a dense plaque on the cytoplasmic face of the membrane. Cadherin molecules form the actual anchor by attaching to the cytoplasmic plaque, extending through the membrane and binding strongly to cadherins coming through the membrane of the adjacent cell.

In order to simulate this microenvironment, Burdick and his collaborators and colleagues used a peptide sequence that mimics these cadherin interactions and bound them to the hydrogels that were then used to encapsulate the MSCs.

According to Mauck, “While the direct link between cadherins and chondrogenesis is not completely understood, what’s known is that if you enhance these interactions early during tissue formation, you can make more cartilage, and, if you block them, you get very poor cartilage formation. What this gel does is trick the cells into think it’s got friends nearby.”

See L Bian, et al., PNAS 2013; DOI:10.1073/pnas.1214100110.

Synthetic Silicate Stimulates Stem Cells to Form Bone Cells


Researchers from Boston, MA have used synthetic silicate nanoplatelest or layered clay to induce bone cell formation from stem cells in the absence of other bone-inducing factors.

Synthetic silicates are composed of either simple or complex salts of silicic acid (SiH4O4).  Silicic acids have been used extensively in commercial and industrial applications that include food additives, glass and ceramic filler materials, and anti-caking agents.

In this study, novel silicate nanoplatelets were constructed that stimulated human mesenchymal stem cells to differentiate into bone-making cells in the absence of any bone-inducing growth factors or cytokines.  The presence of the silicate triggers a set of events inside the mesenchymal stem cells that re-enacts the steps cell normally take during development when they form become bone cells.  These exciting findings illustrate how the use of these silicate nanoplatelets in designing bioactive scaffolds for tissue engineering can lead to the formation of clinically useful bone tissues.

The lead author of this work, Ali Khademhosseini from the division of biomedical engineering at Brigham and Woman’s Hospital, thinks that silicic acid derivatives might be useful in engineering bone. “With an aging population in the U.S., injuries and degenerative conditions are subsequently on the rise,” said Khademhosseini. This means that there is also an increased demand for therapies to repair damaged tissues. Forming such tissues requires protocols to direct stem cell differentiation so that the cells can form new tissues and biomaterials. According to Khademhosseini, “Silicate nanoplatelets have the potential to address this need in medicine and biotechnology.”

“Based on the strong preliminary studies, we believe that these highly bioactive nanoplatelets may be utilized to develop devices such as injectable tissue repair matrixes, bioactive filters, or therapeutic agents for stimulating specific cellular responses in bone-related tissue engineering,” said Akhilesh Gaharwar, first author of this present study.

Future mechanistic studies are necessary to elucidate those underlying pathways that govern the induction of bone differentiation by materials like silicates. Such studies should lead to a better understanding of how particular strategies can be adjusted to improve the performance of lab constructed biomaterials, and accelerate patient recovery time.

Baby from Ohio Saved With An Airway Splint Made by A 3-D Printer


A baby boy from Ohio, Kaiba (KEYE’-buh) Gionfriddo, was born with a trachea (windpipe) that was fragile and kept collapsing. Without precious oxygen, he choked and passed out. Even though the physicians attending him thought about using an airway splint to open his airway, they had yet to implant it. Kaiba was not getting any better, and without a way to get him the oxygen that his little body desperately needed, he did not have much time. All he could do was lie in a hospital bed on a breathing machine.

Kaiba Gionfriddo

To solve this problem, the doctors used plastic particles and a 3-D laser printer to generate an airway splint to deliver oxygen to his lungs. This is a technological first and is the latest advance in the quickly advancing field of regenerative medicine that tries to make human body parts in the lab.

The even more stupendous aspect of this feat is that the production of the tracheal tube only too k one day. Yes, in a single day they “printed out” 100 tiny tubes by employing computer-guided lasers to stack and fuse thin layers of plastic to form various shapes and sizes. The next day, with special permission from the US Food and Drug Administration, they implanted one of these tubes in Kaiba. Needless to say, this is the first time such a treatment has even been done.

Suddenly, Kaiba, whom doctors said would probably never leave the hospital alive, could breathe normally for the first time. Kaiba was 3 months old when the operation was done last year and is nearly 19 months old now. He is about to have his tracheotomy tube removed since it was placed in his throat when he was a couple of months old. He no longer needs a breathing machine and has had not had a single breathing crisis since coming home a year ago.

“He’s a pretty healthy kid right now,” says Dr. Glenn Green, a pediatric ear, nose and throat specialist at C.S. Mott Children’s Hospital of the University of Michigan in Ann Arbor, where the operation was done. This remarkable feat of tissue engineering is described in the New England Journal of Medicine.

Independent experts have highly praised this and the potential 3-D printing provides for creating and quickly manufacturing body parts to solve unmet medical needs.

“It’s the wave of the future,” says Dr. Robert Weatherly, a pediatric specialist at the University of Missouri in Kansas City. “I’m impressed by what they were able to accomplish.”

So far, only a few adults have had trachea, or windpipe transplants, and these are usually used to replace windpipes destroyed by cancer. The windpipes came from dead donors or were lab-made, sometimes using stem cells. Last month, a 2-year-old girl born without a windpipe received one grown from her own stem cells on a plastic scaffold at a hospital in Peoria, Ill.

Kaiba, however, had a different problem; namely an incompletely formed bronchus. The bronchi are the tubes that branch from the windpipe to the lungs. Approximately 2,000 babies are born with such defects each year in the United States and most outgrow them by age 2 or 3, as they grow and mature and their respiratory tract replaces the lost tissues.

In severe cases, parents learn of the defect when the child suddenly stops breathing and dies. That almost happened when Kaiba was 6 weeks old at a restaurant with his parents, April and Bryan Gionfriddo, who live in Youngstown, in northeast Ohio. “He turned blue and stopped breathing on us,” and his father did CPR to revive him, April Gionfriddo says.

More episodes followed, and Kaiba had to go on a breathing machine when he was 2 months old. Doctors told the couple his condition was grave. “Quite a few of them says he had a good chance of not leaving the hospital alive. It was pretty scary,” his mother says. “We pretty much prayed every night, hoping that he would pull through.”

Fortunately a physician at Akron Children’s Hospital named Dr. Marc Nelson suggested the experimental work in Michigan in which researchers were testing airway splints made from biodegradable polyester that are sometimes used to repair bone and cartilage.

Kaiba had the operation on Feb. 9, 2012. The splint was placed around his defective bronchus, which was stitched to the splint to keep it from collapsing. The splint has a slit along its length so it can expand and grow as the child does — something a permanent, artificial implant could not do.

The plastic from which the splint is made is designed to degrade and gradually be absorbed by the body over three years, as healthy tissue forms to replace it, according to the biomedical engineer who led the work, Scott Hollister.

Green and Scott Hollister have a patent pending on the device and Hollister has a financial interest in a company that makes scaffolds for implants.

Dr. John Bent, a pediatric specialist at New York’s Albert Einstein College of Medicine, says only time will tell if this proves to be a permanent solution, but he praised the researchers for persevering to develop it.

“I can think of a handful of children I have seen in the last two decades who suffered greatly … that likely would have benefited from this technology,” Bent says.

Bioengineered Trachea Implanted into a Child


Hannah Genevieve Warren was born in 2010 in Seoul, South Korea with tracheal agenesis, which is to say that she was born without a trachea. Hannah had a tube inserted through her esophagus to her lungs that allowed her to breathe. Children with tracheal agenesis usually die in early childhood, 100% of the time. No child with this condition has ever lived past six years of life. Hannah spent the first two years of her life at the Seoul National Hospital before she was transported to Illinois for an unusual surgery.

While at the Children’s Hospital of Illinois, on April 9, 2013, Hannah had a bioengineered trachea transplanted into her body. This trachea was the result of a remarkable feat of technology called the InBreath tracheal scaffold and bioreactor system that was designed and manufactured by Harvard Bioscience, Inc. Harvard Bioscience, or HBIO, is a global developer, manufacturer and marketer of a broad range of specialized products, primarily apparatus and scientific instruments, used to advance life science research and regenerative medicine.

InBreath tracheal scaffold
InBreath tracheal scaffold

Hannah’s tracheal transplant was the first regenerated trachea transplant surgery that used a biomaterial scaffold that manufactured by the Harvard Apparatus Regenerative Technology (HART) Inc., a wholly owned subsidiary of Harvard Bioscience. HART ensured that the scaffold and bioreactor were custom-made to Hannah’s dimensions. Then the scaffold was seeded with bone marrow cells taken from Hannah’s bone marrow, and the cells were incubated in the bioreactor for two days prior to implantation. Because Hannah’s own cells were used, her body accepted the transplant without the need for immunosuppressive (anti-rejection) drugs.

InBreath Bioreactor
InBreath Bioreactor

The surgeons who participated in this landmark transplant were led by Dr. Paolo Macchiarini of Karolinska University Hospital and Karolinska Institutet in Huddinge, Stockholm and Drs. Mark J. Holterman and Richard Pearl both of Children’s Hospital of Illinois. This surgery was approved by the FDA under an Investigational New Drug (IND) application submitted by Dr. Holterman.

Dr. Mark Holterman, Professor of Surgery and Pediatrics at University of Illinois College of Medicine at Peoria, commented: “The success of this pediatric tracheal implantation would have been impossible without the Harvard Bioscience contribution. Their team of engineers applied their talent and experience to solve the difficult technical challenge of applying regenerative medicine principles in a small child.”

David Green, President of Harvard Bioscience, said: “We would like to congratulate Dr. Macchiarini, Dr. Holterman, Dr. Pearl and their colleagues for accomplishing the world’s first transplant of a regenerated trachea in a child using a synthetic scaffold and giving Hannah a chance at a normal life. We also wish Hannah a full recovery and extend our best wishes to her family.”

Hannah’s surgery is the seventh successful implant of a regenerated trachea in a human using HART technology. Prior successes included the first ever successful regenerated trachea transplant in 2008, the first successful regenerated trachea transplant using a synthetic scaffold in 2011, and the commencement of the first clinical trial of regenerated tracheas in 2012. HART has plans to commence discussions with the FDA and EU regulatory authorities in the near future regarding the clinical pathway necessary to bring this new therapeutic approach to a wider range of patients who are in need of a trachea transplant.